HALIC UNIVERSITY

FACULTY OF ARTS AND SCIENCES

DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS

MBG204(2)
GENETICS LABORATORY

INSTRUCTOR
Dr. Allison Pinar Eronat

EXPERIMENT 3:
DNA ISOLATION

Experiment Date:23/03/2022

Name of the Student:

YMEN MAZHOUD

Submission Date of the Report: 30/03/2022

1. Aim of the experiment:

 This experiment aims to isolate DNA from bananas or strawberries using a buffer.

2. Introduction:
DNA is the hereditary material found in all living organisms. DNA stands
for deoxyribonucleic acid. This is a macromolecule comprised of subunits
of three main parts, each subunit contains a phosphate group, a sugar, and a
nucleotide: adenine, guanine, pyrimidine, and tyrosine, (figure 1). DNA can
be found in a double membrane-bound organelle called the nucleus or freely
floating in a region called the nucleoid. Eukaryotes have linear and
membrane-bound double helix DNA, whereas prokaryotes have one circular
DNA located in the nucleoid region.

Figure 1. DNA monomers and double-helix structure of DNA.[1]

DNA encodes for genes that are responsible for the transcription of
proteins. Those proteins are responsible for all the functions carried out in a
cell. Therefore, DNA is responsible for all cellular mechanisms. Eukaryotic
cells possess mitochondria and/or chloroplasts. These two organelles
contain DNA that is situated freely in the cell. Prokaryotic cells contain
plasmids, and circular DNA, (figure 2) shows both eukaryotic and
prokaryotic DNA, as well as plasmids.
 Figure 2. Comparison between prokaryotic cell (a) and eukaryotic cell (b). [2]

DNA must be extracted and isolated to study the hereditary criteria and
genetics of living organisms. However, eukaryotic DNA is enclosed within
two double membrane-bound layers, the cellular and nuclear membranes.
Therefore, chemical and physical means must be used to break down these
membranes in an attempt to facilitate DNA isolation. Using a buffer
solution is one of the many ways that can be used to disintegrate the
membranes. The detergent in the buffer interacts with the phospholipid
layer, hence, breaking the cell membrane down. The salt’s function is to
neutralize the negative charge of the DNA and decrease its solubility, so a
precipitate is easily formed. Finally, alcohol is added to help the nucleic
acid’s precipitation and separation from the remaining cell fragments. The
extracted DNA can be isolated and stored for further studies.

3. Materials:
Equipment:
- Falcon tubes
- Watch-glass
- Spatula
- Tube holder
- Micropipette
- Graduated cylinder
- 5ml plastic tubes
- Marker
- Ziplock bag
- Mortar and pestle
 - Filter paper
- Beaker
- Funnel
- Wooden swab
Devices:
- Precision balance
- Refrigerator
Biological materials:
- Strawberry
- Banana
Chemicals:
- Distilled water
- NaCl
- NaHCO3
- Detergent
- Ethanol
- Isopropanol

4. Experimental procedure:
a- TE Buffer solution preparation:
i. Weigh 0.38g of sodium chloride (NaCl) and 1.25g of sodium hydrogen
carbonate (NaHCO3) and transfer them into a beaker. Add 30ml of water to
the mixture and stir until the dissolution of substances.
ii. The buffer is cooled at 4 degrees Celsius for about 15 minutes.
b- DNA isolation procedure:
i. About a 4cm long piece of banana is sliced and crushed using a mortar and
pestle. A Ziplock bag can also be used. The banana must be mashed until a
very smooth texture is obtained. In the case of using strawberries, the
piece is dropped in a Ziplock bag and mashed using hands or the bottom
of a beaker.
ii. The cooled buffer solution can be added while crushing the fruit or
afterward.
iii. The buffer and fruit are mixed thoroughly for a few minutes then strained
using filter paper and funneled into a falcon tube.
iv. Once the filtrate is obtained, isopropanol is added gradually and dropwise
to the banana filtrate until two distinguishable layers are seen. The DNA
floats in the upper layer. It has a filamentous structure of white color.
v. If strawberries are used, ethanol is added to the filtrate instead of
isopropanol. The same will be observed with both fruits.
vi. DNA is then isolated by inserting a wooden swab and looping it around
the floating filaments.
vii. The isolated DNA is transferred to a small plastic tube and labeled for
further use.

5. Results:
 Two fruits were used in this experiment, a strawberry, and a banana. It was easier to
extract DNA from strawberries than from bananas. A small quantity of DNA was
observed from the banana sample.

6. Discussion:

in this experiment, we isolated DNA from both bananas and strawberries. Initially, we
crushed the fruits (physical mean) to propagate the cells in clusters. The buffer solution
was used to chemically break down the cell membrane barrier, and the nuclear
membrane. This occurred through the interaction of the detergent with the lipid groups of
the phospholipid bilayer. The weakening of the cell membrane leads to lysis.
The NaCl used in this buffer has the function of neutralizing the charge of DNA. DNA is
a negatively charged molecule. DNA extraction must occur in very low temperatures to
decrease its solubility in water. However, adding alcohol favors the precipitation of DNA
even further. DNA is less soluble in alcohol than in water due to its lack of polarity. The
low temperature of alcohol is another driving factor. [3]
After extracting the DNA, it was stored in a vial and impregnated with TE buffer
solution. TE buffer is slightly basic, it maintains the DNA structure by inactivating
enzymes such as DNase. Distilled water is not a suitable liquid for the long-term storage
of DNA because of its slightly acidic pH. [4]
7. References:
[1] https://en.wikipedia.org/wiki/Base_pair#/media/File:DNA_base-pair_diagram.jpg.
Accessed on 29/03/2023.
[2] https://www.mun.ca/biology/scarr/iGen3_05-09.html Accessed on 29/03/2023.
[3] https://letstalkscience.ca/educational-resources/backgrounders/dna-
extraction#:~:text=DNA%20is%20soluble%20in%20water,form%20a%20visible
%20white%20precipitate. Accessed on 29/03/2023.
[4]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667532/#:~:text=TE%20buffer
%20method%20to%20extract%20DNA%20from%20DBS&text=The%20purpose%20of
%20TE%20buffer,1996). Accessed on 29/03/2023.